CN108243961B - Establishment and rapid breeding method of regeneration system of gentiana wulingensis seeds - Google Patents
Establishment and rapid breeding method of regeneration system of gentiana wulingensis seeds Download PDFInfo
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Abstract
The invention provides a method for establishing a regeneration system and quickly breeding gentiana wulingensis seed, which solves the problems of few wild resources, good medicinal value, weak natural propagation capacity, weak spreading and diffusing capacity, difficult natural renewal, scarcity of excellent seedlings and the like of gentiana wulingensis in the prior art, collects gentiana wulingensis capsule which is full to 80-90% of maturity and is not cracked, puts the gentiana wulingensis capsule into a freshness protection bag for packaging, removes impurities around the capsule after being taken back to a laboratory, uses a detergent for rinsing and sterilization, uses a sterilization filter paper for absorbing surface moisture, longitudinally cuts the fruit by using a scalpel, uses a pair of tweezers for clamping the seed, and uniformly inoculates the seed to a culture medium. Performing bud induction culture and bud multiplication culture; inducing rootless seedlings to root, wherein the rooting rate can reach 100%, the transplanting humidity of the seedlings is kept between 75% and 85%, direct sunlight is avoided, and the survival rate can reach more than 99%; the survival rate of the transplanted seedlings is high, a large amount of cost is saved, the seedlings are strong and straight, and the seedlings grow well and are tidy and consistent.
Description
Technical Field
The invention belongs to the field of plant propagation, and particularly relates to a method for establishing a regeneration system and quickly breeding seeds of gentiana wulingensis.
Background
Gentiana lutea (Gentiana davidii) is also called Lonicera Japonica flos, Gentiana scabra Bunge, Cyprinus Carpio gallbladder, Cyprinus carpioides gallbladder, Gentiana of Gentianaceae, and is 10-20 cm high. The leaf pair is in the shape of rectangular needle or linear needle with the length of 2.5-4 cm and the width of 0.5-1 cm, the base is narrowed and connected, and the vegetative twig is in the shape of rosette. The flowers are clustered into the top of the stem, the head is shaped, and the base is surrounded by 3-5 leaves on the upper part of the stem; the calyx is funnel-shaped, the splinters are not equally large, and the strip shape is like a needle; the corolla is funnel-shaped, purple, split oval, ovary oval and has a handle. Capsule, seed gray brown, nearly circular, surface honeycomb. Grows on hillside grass, hillside roadside, forest edge and under forest, and has the elevation of 350-2500 meters. Produced in Hunan, Jiangxi, Anhui, Zhejiang, Fujian, Guangdong, and Guangxi provinces. Has effects in clearing away heat and toxic materials, promoting urination, and improving eyesight. Used for treating pyogenic osteomyelitis, urinary tract infection, conjunctivitis; it is indicated for furuncle and carbuncle externally.
Disclosure of Invention
The invention aims to provide a method for establishing a regeneration system and quickly breeding gentiana wulingensis seed, which solves the problems of few wild resources, good medicinal value, weak natural propagation capacity and propagation and diffusion capacity, difficult natural updating, scarce fine seedlings and the like of gentiana wulingensis in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for establishing a regeneration system and quickly breeding a gentiana wulingensis seed comprises the following steps:
1) selecting and sterilizing materials: picking 80-90% of mature pods in the current year, rinsing the pods with a detergent, and then placing the pods in running water for washing for 20 minutes; seed disinfection treatment: cleaning pods, soaking in supernatant of saturated bleaching powder for 15min, scrubbing the exterior of the pods with a soft brush, washing, dripping tap water for 0.5-1h, flushing with double distilled water for 2-3 times, sterilizing with 75% alcohol in an ultraclean workbench for 30s, pouring out the alcohol, adding 0.1% mercuric chloride for treatment for 10-13min, pouring mercury liquid into a waste mercury bottle, flushing with sterile water for 3-4 times, and absorbing surface moisture with sterile filter paper for later use;
2) and (3) induction culture: taking the filled and disinfected pods, clamping the pods by using gun-shaped tweezers, splitting the pods by using an operating knife along the vertical direction of the pods, taking out seeds in the pods, and respectively inoculating the seeds in a bud induction culture medium which is sterilized at high temperature and high pressure; the culture conditions are as follows: culturing under the lamplight-free condition for 5-8 days after inoculation, adjusting the illumination intensity to 1000-1500 lx after the lamplight-free culture is finished, culturing for 15-20 days, controlling the temperature of a culture room to be 23 +/-2 ℃, and illuminating for 5h/d to obtain an induced bud plant;
3) and (3) proliferation culture: cutting the obtained bud plant with good growth after the induction culture into stem sections with the length of 1.0-1.5cm, inoculating the stem sections into a propagation culture medium, and performing propagation culture for 10h/d, 1500-2000lx and 20-30d to obtain seedlings subjected to propagation culture;
4) inducing and rooting: cutting off a single rootless seedling plant with 2-3 leaves and a plant height of 3-4cm after propagation culture, and transferring the cut rootless seedling plant into a rooting culture medium; the illumination time is 10h/d, the illumination intensity is 1500-;
5) and (3) completing the test-tube plantlet culture: when the induced rooting culture test-tube plantlet grows to 5-6cm high, the roots are 3-5, the leaves are 5, and the length of the leaves is 2-3cm, the induced germination and rapid micropropagation culture of the gentiana wulingensis seed are completed;
6) after the test-tube plantlet after rooting culture is put in natural light for hardening for 5-7 days, the bottle cap is opened for hardening for 1-2 days so as to enhance the adaptability of the test-tube plantlet to the outdoor environment; then taking out the cultured plant from the culture bottle, washing off the residual culture medium attached to the root system, transferring into a matrix mixed by vermiculite and humus soil according to the mass ratio of 1:2, preserving moisture and shading, controlling the temperature at 15-30 ℃, keeping the humidity at 75-85%, and avoiding direct sunlight; after 2-3 weeks, the plant adaptability is gradually enhanced, and healthy and complete seedlings which naturally grow are obtained.
The bud induction culture medium is N6+1.5mg/L6-BA +0.5mg/LKT +0.3mg/LNAA +20g/L sucrose +5.5g/L agar powder +1.0 g.L-1Activated carbon; the pH was 5.8.
The proliferation culture medium is N6+2.0 mg/L6-BA +0.5mg/L KT +0.1mg/L NAA +20g/L sucrose +5.5g/L agar powder +1.0 g.L-1Activated carbon; the pH was 5.8.
The rooting culture medium is 1/2N6, 1.0mg/L IBA, 0.1mg/L NAA, 25g/L sucrose, 6.5g/L agar powder and 1.5 g.L-1Activated carbon; the pH was 5.8.
The invention has the advantages that:
compared with the prior art, the invention has the advantages and beneficial effects that 1, the selection of the system establishing material is as follows: collecting uncracked Wuling gentian capsule with the full maturity of 80-90%; 2. proper sterilization time and material handling; 3. accurate and reasonable culture medium composition; 4. the reasonable setting of the illumination intensity in the illumination time improves the rooting rate of the induced rootless seedlings, the rooting rate can reach 100 percent, and the survival rate can reach more than 99 percent; the survival rate of the transplanted seedlings is high, the plant diseases and insect pests are few, a large amount of cost is saved, the seedlings are strong and tall, and the growth vigor is good and uniform.
Drawings
FIG. 1 shows a shoot plant of a seed after induction culture.
FIG. 2 proliferation culture.
FIG. 3 growth in enrichment culture.
FIG. 4 strong seedling culture.
FIG. 5 shows seedlings cultured by induced rooting.
FIG. 6 rooting of plants.
And fig. 7 transplanting planting.
Detailed Description
Example 1
1) Selecting and sterilizing materials: picking up 85% of ripe pods in the current year, rinsing the pods with a detergent, and then placing the pods in running water for washing for 20 minutes; seed disinfection treatment: cleaning pods, soaking in the supernatant of saturated bleaching powder for 15min, scrubbing the exterior of the pods with a soft brush, washing, dripping tap water for 1h, flushing with double distilled water for 3 times, placing in an ultraclean workbench, disinfecting with 75% alcohol for 30s, pouring out the alcohol, adding 0.1% mercuric chloride for treatment for 12min, pouring mercury liquid into a waste mercury bottle, flushing with sterile water for 4 times, and absorbing surface moisture with disinfection filter paper for later use;
2) and (3) induction culture: taking the filled and disinfected pods, clamping the pods by using gun-shaped tweezers, splitting the pods by using an operating knife along the vertical direction of the pods, taking out seeds in the pods, and respectively inoculating the seeds in a bud induction culture medium which is sterilized at high temperature and high pressure; the culture conditions are as follows: culturing under the lamplight-free condition 7d after inoculation, adjusting the illumination intensity to 1200lx after the lamplight-free culture is finished, culturing for 18d, controlling the temperature of a culture room to be 23 +/-2 ℃ and the illumination time to be 5h/d, and obtaining an induced bud plant;
3) and (3) proliferation culture: cutting the obtained bud plant with good growth after the induction culture into stem segments with the length of 1.5cm, inoculating the stem segments into a multiplication culture medium, and performing multiplication culture for 10h/d, 1800lx and 25d to obtain a seedling for multiplication culture;
4) inducing and rooting: cutting off a single rootless seedling plant with 3 leaves and a plant height of 4cm after propagation culture, and transferring the cut rootless seedling plant into a rooting culture medium; the illumination time is 10h/d, the illumination intensity is 2000lx, and the induced rooting time is 20d, so as to obtain seedlings induced to root; the rooting rate can reach 100 percent;
5) and (3) completing the test-tube plantlet culture: when the induced rooting culture test-tube plantlet grows to 6cm high, 5 roots and 5 leaves are obtained, and the leaf length is 3cm, the induced germination and rapid micropropagation culture of the gentiana wuliang seeds are completed;
6) after the test-tube plantlet after rooting culture is put in natural light for hardening for 6 days, the bottle cap is opened for hardening for 2 days so as to enhance the adaptability of the test-tube plantlet to the outdoor environment; then taking out the cultured plant from the culture bottle, washing off the residual culture medium attached to the root system, transferring into a matrix mixed by vermiculite and humus soil according to the mass ratio of 1:2, preserving moisture and shading, controlling the temperature at 25 ℃, keeping the humidity at 80% and avoiding direct sunlight; after 3 weeks, the plant adaptability is gradually enhanced, and healthy and complete seedlings which naturally grow are obtained.
The bud induction culture medium is N6+1.5mg/L6-BA +0.5mg/LKT +0.3mg/LNAA +20g/L sucrose +5.5g/L agar powder +1.0 g.L-1Activated carbon; the pH was 5.8.
The proliferation culture medium is N6+2.0 mg/L6-BA +0.5mg/L KT +0.1mg/L NAA +20g/L sucrose +5.5g/L agar powder +1.0 g.L-1Activated carbon; the pH was 5.8.
The rooting culture medium is 1/2N6, 1.0mg/L IBA, 0.1mg/L NAA, 25g/L sucrose, 6.5g/L agar powder and 1.5 g.L-1Activated carbon; the pH was 5.8.
The survival rate can reach 100 percent.
Example 2
1) Selecting and sterilizing materials: picking 80% of mature pods in the current year, rinsing the pods with a detergent, and then placing the pods in running water for washing for 20 minutes; seed disinfection treatment: cleaning pods, soaking in the supernatant of saturated bleaching powder for 15min, scrubbing the exterior of the pods with a soft brush, washing, dripping tap water for 0.5h, flushing with double distilled water for 2 times, placing in an ultraclean workbench, disinfecting with 75% alcohol for 30s, pouring out the alcohol, adding 0.1% mercuric chloride, treating for 10min, pouring mercury liquid into a waste mercury bottle, flushing with sterile water for 3 times, and absorbing surface moisture with sterile filter paper for later use;
2) and (3) induction culture: taking the filled and disinfected pods, clamping the pods by using gun-shaped tweezers, splitting the pods by using an operating knife along the vertical direction of the pods, taking out seeds in the pods, and respectively inoculating the seeds in a bud induction culture medium which is sterilized at high temperature and high pressure; the culture conditions are as follows: culturing under the condition of no light 5 days after inoculation, adjusting the illumination intensity to 1000lx after the culture under the condition of no light is finished, culturing for 15 days, controlling the temperature of a culture room to be 23 +/-2 ℃ and the illumination time to be 5h/d, and obtaining an induced bud plant;
3) and (3) proliferation culture: cutting the obtained bud plant with good growth after the induction culture into stem segments with the length of 1.0cm, inoculating the stem segments into a multiplication culture medium, and performing multiplication culture for 10h/d, 1500lx and 20d to obtain a seedling after the multiplication culture;
4) inducing and rooting: cutting off a single rootless seedling plant with 2 leaves and a plant height of 3cm after propagation culture, and transferring the cut rootless seedling plant into a rooting culture medium; the illumination time is 10h/d, the illumination intensity is 1500lx, and the induced rooting time is 15d, so as to obtain seedlings induced to root; the rooting rate can reach 100 percent;
5) and (3) completing the test-tube plantlet culture: when the induced rooting culture test-tube plantlet grows to the height of 5cm, 3 roots and 5 leaves are obtained, and the leaf length is 2cm, the induced germination and rapid micropropagation culture of the gentiana wulingensis seed are completed;
6) after the test-tube plantlet after rooting culture is put in natural light for hardening for 5 days, the bottle cap is opened for hardening for 1 day to enhance the adaptability of the test-tube plantlet to the outdoor environment; then taking out the cultured plant from the culture bottle, washing off the residual culture medium attached to the root system, transferring into a matrix mixed by vermiculite and humus soil according to the mass ratio of 1:2, preserving moisture and shading, controlling the temperature at 15 ℃, keeping the humidity at 75% and avoiding direct sunlight; after 2 weeks, the plant adaptability is gradually enhanced, and healthy and complete seedlings which naturally grow are obtained.
The bud induction culture medium is N6+1.5mg/L6-BA +0.5mg/LKT +0.3mg/LNAA +20g/L sucrose +5.5g/L agar powder +1.0 g.L-1Activated carbon; the pH was 5.8.
The proliferation culture medium is N6+2.0 mg/L6-BA +0.5mg/L KT +0.1mg/L NAA +20g/L sucrose +5.5g/L agar powder +1.0 g.L-1Activated carbon; the pH was 5.8.
The rooting culture medium is 1/2N6, 1.0mg/L IBA, 0.1mg/L NAA, 25g/L sucrose, 6.5g/L agar powder and 1.5 g.L-1Activated carbon; the pH was 5.8.
The survival rate can reach 99%.
Example 3
1) Selecting and sterilizing materials: picking up 90% of mature pods in the current year, rinsing the pods with a detergent, and then placing the pods in running water for washing for 20 minutes; seed disinfection treatment: cleaning pods, soaking in the supernatant of saturated bleaching powder for 15min, scrubbing the exterior of the pods with a soft brush, washing, dripping tap water for 1h, flushing with double distilled water for 2-3 times, sterilizing in an ultraclean workbench with 75% alcohol for 30s, pouring out the alcohol, adding 0.1% mercuric chloride for treatment for 13min, pouring mercury liquid into a waste mercury bottle, flushing with sterile water for 4 times, and absorbing surface moisture with sterile filter paper for later use;
2) and (3) induction culture: taking the filled and disinfected pods, clamping the pods by using gun-shaped tweezers, splitting the pods by using an operating knife along the vertical direction of the pods, taking out seeds in the pods, and respectively inoculating the seeds in a bud induction culture medium which is sterilized at high temperature and high pressure; the culture conditions are as follows: culturing under a lighting-free condition for 8 days after inoculation, adjusting the illumination intensity to 1500lx after the lighting-free culture is finished, culturing for 20 days, controlling the temperature of a culture room to be 23 +/-2 ℃, and illuminating for 5h/d to obtain an induced bud plant;
3) and (3) proliferation culture: cutting the obtained bud plant with good growth after the induction culture into stem segments with the length of 1.5cm, inoculating the stem segments into a multiplication culture medium, and performing multiplication culture for 10h/d, 2000lx and 30d to obtain a seedling subjected to the multiplication culture;
4) inducing and rooting: cutting off a single rootless seedling plant with 3 leaves and a plant height of 4cm after propagation culture, and transferring the cut rootless seedling plant into a rooting culture medium; the illumination time is 10h/d, the illumination intensity is 2000lx, and the induced rooting time is 20d, so as to obtain seedlings induced to root; the rooting rate can reach 100 percent;
5) and (3) completing the test-tube plantlet culture: when the induced rooting culture test-tube plantlet grows to 6cm high, 5 roots and 5 leaves are obtained, and the leaf length is 3cm, the induced germination and rapid micropropagation culture of the gentiana wuliang seeds are completed;
6) after the test-tube plantlet after rooting culture is put in natural light for 7 days of acclimatization, the bottle cap is opened for acclimatization for 2 days so as to enhance the adaptability of the test-tube plantlet to the outdoor environment; then taking out the cultured plant from the culture bottle, washing off the residual culture medium attached to the root system, transferring into a matrix mixed by vermiculite and humus soil according to the mass ratio of 1:2, preserving moisture and shading, controlling the temperature at 30 ℃, keeping the humidity at 85 percent, and avoiding direct sunlight; after 3 weeks, the plant adaptability is gradually enhanced, and healthy and complete seedlings which naturally grow are obtained.
The bud induction culture medium is N6+1.5mg/L6-BA +0.5mg/LKT +0.3mg/LNAA +20g/L sugarcaneSugar +5.5g/L agar powder +1.0 g.L-1Activated carbon; the pH was 5.8.
The proliferation culture medium is N6+2.0 mg/L6-BA +0.5mg/L KT +0.1mg/L NAA +20g/L sucrose +5.5g/L agar powder +1.0 g.L-1Activated carbon; the pH was 5.8.
The rooting culture medium is 1/2N6, 1.0mg/L IBA, 0.1mg/L NAA, 25g/L sucrose, 6.5g/L agar powder and 1.5 g.L-1Activated carbon; the pH was 5.8.
The survival rate can reach 100 percent.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (1)
1. A method for establishing a regeneration system and quickly breeding a gentiana wulingensis seed is characterized by comprising the following steps: the method comprises the following steps:
1) selecting and sterilizing materials: picking 80-90% of mature pods in the current year, rinsing the pods with a detergent, and then placing the pods in running water for washing for 20 minutes; seed disinfection treatment: cleaning pods, soaking in supernatant of saturated bleaching powder for 15min, scrubbing the exterior of the pods with a soft brush, washing, dripping tap water for 0.5-1h, flushing with double distilled water for 2-3 times, sterilizing with 75% alcohol in an ultraclean workbench for 30s, pouring out the alcohol, adding 0.1% mercuric chloride for treatment for 10-13min, pouring mercury liquid into a waste mercury bottle, flushing with sterile water for 3-4 times, and absorbing surface moisture with sterile filter paper for later use;
2) and (3) induction culture: taking the filled and disinfected pods, clamping the pods by using gun-shaped tweezers, splitting the pods by using an operating knife along the vertical direction of the pods, taking out seeds in the pods, and respectively inoculating the seeds in a bud induction culture medium which is sterilized at high temperature and high pressure; the culture conditions are as follows: culturing under the lamplight-free condition for 5-8 days after inoculation, adjusting the illumination intensity to 1000-1500 lx after the lamplight-free culture is finished, controlling the temperature of a culture room to be 23 +/-2 ℃, the illumination time to be 5h/d, and the culture time to be 15-20 days to obtain an induced bud plant;
3) and (3) proliferation culture: cutting the obtained bud plant with good growth after the induction culture into stem sections with the length of 1.0-1.5cm, inoculating the stem sections into a propagation culture medium, and performing propagation culture for 10h/d, 1500-2000lx and 20-30d to obtain seedlings subjected to propagation culture;
4) inducing and rooting: cutting off a single rootless seedling plant with 2-3 leaves and a plant height of 3-4cm after propagation culture, and transferring the cut rootless seedling plant into a rooting culture medium; the illumination time is 10h/d, the illumination intensity is 1500-;
5) and (3) completing the test-tube plantlet culture: when the induced rooting culture test-tube plantlet grows to 5-6cm high, the roots are 3-5, the leaves are 5, and the length of the leaves is 2-3cm, the induced germination and rapid micropropagation culture of the gentiana wulingensis seed are completed;
6) after the test-tube plantlet after rooting culture is put in natural light for hardening for 5-7 days, the bottle cap is opened for hardening for 1-2 days so as to enhance the adaptability of the test-tube plantlet to the outdoor environment; then taking out the cultured plant from the culture bottle, washing off the residual culture medium attached to the root system, transferring into a matrix mixed by vermiculite and humus soil according to the mass ratio of 1:2, preserving moisture and shading, controlling the temperature at 15-30 ℃, keeping the humidity at 75-85%, and avoiding direct sunlight; after 2-3 weeks, the plant adaptability is gradually enhanced, and healthy seedlings are obtained;
the bud induction culture medium is N6+1.5mg/L6-BA +0.5mg/LKT +0.3mg/LNAA +20g/L sucrose +5.5g/L agar powder +1.0 g.L-1Activated carbon; the pH value is 5.8;
the proliferation culture medium is N6+2.0mg/L of 6-BA +0.5mg/L of KT +0.1mg/L of NAA +20g/L of sucrose +5.5g/L of agar powder +1.0 g.L-1Activated carbon; the pH value is 5.8;
the rooting culture medium is 1/2N6+1.0mg/L IBA +0.1mg/L NAA +25g/L sucrose +6.5g/L agar powder +1.5 g.L-1Activated carbon; the pH was 5.8.
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